Double Beam spectrometer

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Double Beam spectrometer

• Provides a signal that is largely free of drift
  in the source and detector without requiring
  really expensive components
• Beam alternates very fast between sample and
  reference cells.
• Dont need to keep zeroing

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Sample Cells

• Usually 1 cm pathlength
• Glass visible
• Quartz UV
• Plastic disposable beware solvents
• 5 cm or 10 cm for dilute samples
• Smaller cells for small samples
• Flow through cells
• Temperature control
• Gas cells longer
• Fibre optic probes

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Fibre Optics

• Fibres of glass, usually about 120 µm in
  diameter.

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Fibre Optic Probe
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Derivative Spectroscopy

• Can determine flat maxima more precisely
• Isolate shoulders
• Distinguish weak signals from background

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Photometric Titration
Solution 2 x 10-3 M in Bi3 and Cu2 Titrant
EDTA At 745 nm, neither cation, nor reagent,
absorbs Bi complex forms first more stable
but doesnt absorb The cu complex does absorb at
745 nm
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Reaction rates

• Following enzyme kinetics
• Determine enzymes
• Determine substrates

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Stop Flow Methods

• For fast reactions
• Two syringes driven at same rate
• Solutions flow into mixing chamber
• When plunger hits stop, measurement starts
• Generally measure initial rates of reactions

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Absorbance ratios and differences

• Measure A at two ?
• Use A1/A2 or A1 A 2
• Plot versus concentration
• Use to asses purity of samples to check just
  one component is present
• Eg ratio of A 260 nm A 280 nm indicates how pure
  A sample of DNA you have.

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Applications

• Metal ion analysis
• eg iron II or III
• React with ligands to get intense colours
• Reduce Fe III with hydroxylamine or hydroquinone
  etc
• Can extract the complexes into isoamyl alcohol
  for a cleanup/preconcentration step
• 0.1 - 0.005 µg/mL are typical LODs

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Organic/Biologicals

• Most common application
• Many absorb strongly
• May need to derivatize
• eg alcohol with phenyl isocyanate to give alkyl
  carbamates 280 nm
• Free amino acids react with ninhydrin
  blue/purple 575 nm aa analyzers

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Automated clinical methods

• Many samples/hour
• Expensive to buy
• Can run many samples unattended

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Centrifugal Analysis

• Combines robotic pipettors
• Centrifuge
• Spectrophotometer
• Computer
• Increases sample throughput
• Reduces volume of sample and reagents

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• Eliminates chemistry changeover time
• No set-up equilibrium time
• Used for water quality measurements
• Based on standard procedures
• Liquids are dispensed into separate compartments
  attached to the cuvettes
• Cuvettes are round a rotor. When rotor is spun,
  reagents are propelled into cuvettes.
• All reactions start together good for kinetics

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• Samples and standards are mixed and run in
  parallel.
• Identical conditions are ensured for all cuvettes
• Can analyze 110 samples/hour
• Rotor spins at 2000 revolutions /min
• Get an average of 7 readings/sample

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Water Pollution Analysis

• Molybdenum blue method
• (NH4)3 P (Mo3O10)4 yellow
• Reduce with hydroquinone, Sn II or Fe II
• Get a polymer of Mo of different oxidation states
• Not stoichiometrically well-defined but is blue
• As, Si interfere so they can also be determined
  this way

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Air Pollution Analysis- SO2

• Collect by bubbling through 0.1 M sodium
  tetrachloromercurate
• HgCl42- 2SO2 2H2O ? Hg(SO3)22-
  4Cl- 4H
• Treat with formaldehyde and p-rosaniline to give
  red-violet colour 569 nm
• 0.005 ppm by volume
• NO2 interferes above 2 ppm

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Advantages Visible

• Less interferences
• Often higher molar absorptivities